Use of n-alkyl imidazole for sulfurization of oligonucleotides with an acetyl disulfide

ABSTRACT

A method and compositions for sulfurizing at least one phosphite or thiophosphite linkage in an oligonucleotide. The addition of N-alkyl imidazole to the acetyldisulfide sulfurization solution enables the use of industrially preferred solvents or solvents that are derived from renewable resources.

FIELD

Methods and compositions for synthesizing sulfurized oligonucleotidesare disclosed.

INTRODUCTION

Modified oligonucleotides find use in a variety of applications,including research, diagnostic and therapeutic applications. Inparticular, with respect to therapeutic applications, the modifiedoligonucleotides find use as any DNA, RNA or other nucleic acidtherapeutic, such as antisense nucleic acids, in gene therapyapplications, aptamers and more recently interfering RNA (i.e., iRNA orRNAi) applications, etc.

Phosphorothioate analogues, in particular, are of considerable interestin nucleic acid research, diagnostics and therapeutics (Eckstein, F. andGish, G. (1989) TIBS, 14, 97-100). The substitution of a sulfur atom fora non-bridging oxygen atom significantly changes the ability of theinternucleotide bond to be degraded by cellular nucleases (Zon, G. andStec, W. J. (1991) In Eckstein, F. (ed.), Oligonucleotides andAnalogues: A Practical Approach. IRL Press, Oxford, pp. 87-108.)

Introduction of phosphorothioate moieties into oligonucleotides,assembled by solid-phase synthesis, can be achieved readily in two ways.The H-phosphonate approach involves a single sulfur transfer step,carried out after the desired sequence has been assembled, to convertall of the internucleotide linkages to phosphorothioates (Agrawal, S.and Tang, J.-Y. Tetrahedron Lett., 31, (1990) 7541-7544).

Alternatively, the phosphoramidite approach (Matteucci, M. D.,Caruthers, M. H. J. Am. Chem. Soc., 103, (1981), 3186-3191) features achoice at each synthetic cycle: a standard oxidation using iodine andwater provides the normal phosphodiester internucleotide linkage,whereas a sulfurization step introduces a phosphorothioate at thatspecific position in the sequence (Stec, W. J., Zon, G., Egan, W. andStec, B., J. Am. Chem. Soc., 106, (1984), 6077-6079). An advantage ofusing phosphoramidite chemistry, therefore, is the capability to controlthe state of each linkage [P═O versus P═S] in a site-specific manner.

Phenylacetyl disulfide (PADS) was shown to be an effective sulfurtransfer reagent and its rate of sulfurization appeared rapid (5minutes) similar to the 3-H-12-benzodithiol-3-one 1,1-dioxide, known asthe “Beaucage Reagent” (Kamer, P. C. J., Roelen, H. C. P. F, van denElst, H., Van Der Marel, G. A, and Van Boom J. H., Tetrahedron Lett.,30, (1989) 6757-6760). However, most published reports of its use werein chlorinated solvents with moderate to high dielectric constants suchas dichloroethane; which are non-preferred for large, industrial-scalesynthesis due to the environmental concerns of exposure and disposal ofhaloalkane solvents. The use of PADS reagent for sulfurization ofnucleotides and oligonucleotides in a mixture of dichloroethane,acetonitrile, and pyridine was described in a 1991 Dutch patentapplication (NL8902521 (A); Production of phosphorothioate ester—byreacting phosphite ester with acyl disulphide, especially in synthesisof nucleic acid analogues). In this patent application the use of PADSwas demonstrated for sulfurization of mononucleotides, dinucleotides andshort oligonucleotides (6 and 7 nucleotides in length). Subsequently,the use of PADS for the synthesis of an oligonucleotide, 20 nucleotidesin length, was demonstrated using a mixture of dichloroethane andcollidine (Wyrzykiewicz, T. K., and Ravikumar, V. T., Bioorganic &Medicinal Chemistry Letters (1994), 4(12), 1519-22). Following thisaccount, it was reported that, similar to tetraethylthiuram disulfide(TETD), PADS was more effective as a sulfur transfer reagent whendissolved in higher dielectric constant solvents such as acetonitrile(Cheruvallath, Z. S.; Wheeler, P. D.; Cole, D. L.; Ravikumar, V. T.,Nucleosides, Nucleotides and Nucleic Acids, (1999) 18, 485-492;Synthesis of Sulfurized Oligonucleotides: U.S. Pat. No. 7,378,516 B2,May 2008, D. L. Cole, V. T. Ravikumar, Z. S. Cheruvallath). It has alsobeen recently disclosed that PADS can be used in 50/50:v/v mixtures of3-picoline with a variety of organic co-solvents such as acetonitrile,toluene, 1-methylpyrrolidinone, and tetrahydrofuran (PADS/NMP forsulfurization of oligonucleotides, IP.com Journal (2005), 5(4), pp69-70).

Acetonitrile is a by-product from the manufacture of acrylonitrile(Peter Pollak, Gérard Romeder, Ferdinand Hagedorn, Heinz-Peter Gelbke“Nitriles” in Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH,2002: Weinheim). Production trends for acetonitrile thus generallyfollow those of acrylonitrile. Acetonitrile can also be produced by manyother methods, but these are currently of no commercial importance andare not used to produce commercial amounts of acetonitrile. As ofOctober 2008, there has been a decrease of acetonitrile production dueto decreased production of acrylonitrile (Chemical & Engineering News,(2008) 86:47, p. 27). . The shortage arose from a lower output fromChina and a U.S. factory in Texas damaged during Hurricane Ike. Theglobal economic slowdown of 2007, 2008, and 2009 has affected the demandand the production of acrylonitrile which is used in the manufacture ofacrylic fibers and acrylonitrile-butadiene (ABS) resins. Along with thelack of availability, the price of acetonitrile has significantlyincreased.

In the community of Large Scale Oligonucleotide Manufacturing, PADS isfound to be an efficient sulfurization reagent available at a reasonablecost, but is taught to be preferably used in acetonitrile containingsolution (Isis U.S. Pat. No. 6,114,519, U.S. Pat. No. 6,242,591, U.S.Pat. No. 7,227,015, U.S. Pat. No. 7,378,516). In the last two years,however, the industrial decrease in acetonitrile production and itsassociated cost increase have prompted the Oligonucleotide Manufacturingcommunity to look at alternative solvents and conditions to use PADS.

SUMMARY

Aspects of this invention include new compositions and methods toproduce phosphorothioate or phosphorodithioate linkages containingoligonucleotides.

DEFINITIONS

Prior to describing embodiments of the invention in further detail, theterms used in this application are defined as follows unless otherwiseindicated.

An “acetyl dislufide” has the formula R²⁰—C(O)—S—S—C(O)—R²¹ where R²⁰and R²¹ may be the same or different substituents, such as hydrocarbyl(for example, C1 to C12 alkyl or aryl, such as phenyl or napthyl), anyof which may be substituted or unsubstituted (for example, with loweralkyl, halo, amino or the like). When R²⁰ and R²¹ are both phenyl, thecompound is phenyl acetyl disulfide.

The term “alkyl” as used herein, unless otherwise specified, refers to asaturated straight chain, branched or cyclic hydrocarbon group of 1 to24, typically 1-12, carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term “lower alkyl” intendsan alkyl group of one to six carbon atoms, and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term“cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “aryl” refers to 5-, 6-, and 7-membered single-ring aromaticgroups that may include from zero to four heteroatoms, for example,benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like. Those aryl groups having heteroatoms in the ring structure mayalso be referred to as “aryl heterocycles” or “heteroaromatics.” Theterm “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (the rings are “fused rings”) wherein at least one of the rings isaromatic (e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, and/or heterocycles). A “loweraryl” contains up to 18 carbons, such as up to 14, 12, 10, 8 or 6carbons.

The term “hydrocarbyl” refers to alkyl, alkenyl or alkynyl. The term“substituted hydrocarbyl” refers to hydrocarbyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents may include, for example, ahydroxyl, a halogen, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate,a phosphinate, an amino, an amido, an amidine, an imine, a cyano, anitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclic, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain may themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CN, and the like. Cycloalkyls may be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CN, and the like.

A “nucleotide” or “nucleotide moiety” refers to a sub-unit of a nucleicacid (whether DNA or RNA or analogue thereof) which includes a phosphategroup, a sugar group and a heterocyclic base, as well as analogs of suchsub-units. Other groups (e.g., protecting groups) can be attached to anycomponent(s) of a nucleotide.

A “nucleoside” or “nucleoside moiety” references a nucleic acid subunitincluding a sugar group and a heterocyclic base, as well as analogs ofsuch sub-units. Other groups (e.g., protecting groups) can be attachedto any component(s) of a nucleoside.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties that contain not only the known purine and pyrimidine bases,e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U),but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, alkylated riboses or other heterocycles. Suchmodifications include, e.g., diaminopurine and its derivatives, inosineand its derivatives, alkylated purines or pyrimidines, acylated purinesor pyrimidines thiolated purines or pyrimidines, and the like, or theaddition of a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl,phenoxyacetyl, dimethylformamidine, dibutylformamidine,dimethylacetamidine, N,N-diphenyl carbamate, or the like. The purine orpyrimidine base may also be an analog of the foregoing; suitable analogswill be known to those skilled in the art and are described in thepertinent texts and literature. Common analogs include, but are notlimited to, 1-methyladenine, 2-methyladenine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

In addition, the terms “nucleoside” and “nucleotide” include thosemoieties that contain not only conventional ribose and deoxyribosesugars and conventional stereoisomers, but other sugars as well,including L enantiomers and alpha anomers. Modified nucleosides ornucleotides also include modifications on the sugar moiety, e.g.,wherein one or more of the hydroxyl groups are replaced with halogenatoms or aliphatic groups, or are functionalized as ethers, amines, orthe like. “Analogues” refer to molecules having structural features suchthat they can be considered mimetics, derivatives, having analogousstructures, or the like, and include, for example, polynucleotides oroligonucleotides incorporating non-natural (not usually occurring innature) nucleotides, unnatural nucleotide mimetics such as 2′-modifiednucleosides including but not limited to 2′-fluoro, 2′-O-alkyl,O-alkylamino, O alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl,O-alkyl imidazole, and polyethers of the formula (O-alkyl)m such aslinear and cyclic polyethylene glycols (PEGs), and (PEG)-containinggroups, locked nucleic acids (LNA), peptide nucleic acids (PNA),oligomeric nucleoside phosphonates, and any polynucleotide that hasadded substituent groups, such as protecting groups or linking groups.

An “internucleotide linkage” or “nucleotide bond” refers to a chemicallinkage between two nucleoside moieties, such as the phosphodiesterlinkage in nucleic acids found in nature or their thiolated ordithiolated equivalents, or linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may include a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g., a sulfur atom, or the nitrogen atom of a mono- ordi-alkyl amino group.

An “N-alkyl imidazole” can include lower N-alkyl imidazoles. In thiscontext “lower” means C1 to C6 in total, unsubstituted or substituted.

A “phospho” group includes a phosphodiester, phosphotriester, andH-phosphonate groups. In the case of either a phospho or phosphitegroup, a chemical moiety other than a substituted 5-membered furyl ringmay be attached to O of the phospho or phosphite group which linksbetween the furyl ring and the P atom.

“Phosphite” is a compound which includes a P(OR)₃, where R is a groupmany of which are known, and includes structures that may be relatedsuch as by disproportionation. A “phoshite” then includes anH-phosphonate, namely HP(O)(OR)₂.

The term “phosphoramidite group” refers to a group comprising thestructure —P—(OR¹³)(NR¹⁴R¹⁵)—, wherein each of R¹³, R¹⁴, and R¹⁵ isindependently a hydrocarbyl, substituted hydrocarbyl, heterocycle,substituted heterocycle, aryl or substituted aryl. In some embodiments,R¹³, R¹⁴, and R¹⁵ may be independently selected from aryls, alkyls, anyof which may be substituted or unsubstituted. Any of R¹³, R¹⁴, or R¹⁵may, for example, include structures containing up to 18, 16, 14, 12,11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 carbons. In some embodiments, R¹³ is2-cyanoethyl or methyl, and either or both of R¹⁴ and R¹⁵ is isopropyl.R¹⁴ and R¹⁵ can optionally be cyclically connected.

The term “phosphothioamidite group” refers to a group comprising thestructure —P—(SR¹³)(NR¹⁴R¹⁵), wherein each of R¹³, R¹⁴, and R¹⁵ isindependently selected from any of those groups already mentioned forR¹³, R¹⁴, and R¹⁵.

The term “Phosphorohioate” usually refers to an analogue of aphosphodiester or phosphotriester linkage in which a non-bridging oxygenhas been replaced by a sulfur atom comprising the structureO—P(S)(OR)—O— or —O—P(S)(O⁻)—O—, wherein R is a substituent such as asubstituted or unsubstituted alkyl or aryl group.

The term “Phosphorodithioate” refers to an analogue of phosphodiester orphosphotriester linkage in which both of the non-bridging oxygen havebeen replaced by a sulfur atom comprising the structure —O—P(S)(SR)—O—or —O—P(S)(S⁻)—O—, wherein R is a substituent such as a substituted orunsubstituted alkyl or aryl group.

An “oligonucleotide”, “polynucleotide” or a “nucleic acid” refers to acompound containing a plurality of nucleoside moiety subunits ornucleoside residues that are linked by internucleotide bonds. As such italso refers to a compound containing 2′-deoxynucleotide orribonucleotide, or nucleotide analogue subunits or mixture thereof.Oligonucleotides may typically have more than 2, 10, 20, or 30nucleotides up to any number of nucleotides (for example, up to 10, 20,40, 60, 80, 100, or 200 nucleotides).

“Linkage” as used herein refers to a first moiety bonded to two othermoieties, wherein the two other moieties are linked via the firstmoiety. Typical linkages include ether (—O—), oxo (—C(O)—), amino(—NH—), amido (—N—C(O)—), thio (—S—), phospho (—P—), ester (—O—C(O)—).

“Low dielectric constant” means a dielectric constant (measured at 20°C.) which is no more or less than 35, and could even be no more or lessthan 32, 25, 20, 15, 10, or 1. A “low dielectric constant solvent” is asolvent with a low dielectric constant.

“High dielectric constant solvent” references a solvent having adielectric constant of greater than 35 as measured at 20° C. including37 or greater, or even greater than 40. High dielectric constantsolvents are preferably aprotic solvents.

A “solvent” can be made up of a single solvent or multiple solvents.Sometimes “solvent” is used interchangeably herein within “solventsystem”.

The term “substituted” as used to describe chemical structures, groups,or moieties, refers to the structure, group, or moiety comprising one ormore substituents. As used herein, in cases in which a first group is“substituted with” a second group, the second group is attached to thefirst group whereby a moiety of the first group (in some cases ahydrogen) is replaced by the second group. “Substituent” references agroup that replaces another group in a chemical structure. In some casessubstituents include nonhydrogen atoms (e.g. halogens), functionalgroups (such as, but not limited to amino, amido, sulfhydryl, carbonyl,hydroxyl, alkoxy, carboxyl, silyl, silyloxy, phosphate and the like),hydrocarbyl groups, and hydrocarbyl groups substituted with one or moreheteroatoms.

Hyphens, or dashes are used at various points throughout thisspecification to indicate attachment, e.g. where two named groups areimmediately adjacent to a dash in the text, this indicates that the twonamed groups area attached to each other. Similarly, a series of namedgroups with dashes between each of the named groups in the textindicated the named groups are attached to each other in the ordershown. Also, a single named group adjacent a dash in the text indicatesthat the named group is typically attached to some other, unnamed group.The attachment indicated by a dash generally represents a covalent bondbetween the adjacent named groups. At various points throughout thespecification, a group may be set forth in the text with or without anadjacent dash, (e.g. amido or amido-, further e.g. alkyl or alkyl-, yetfurther Lnk, Lnk- or -Lnk-) where the context indicates the group isintended to be (or has the potential to be) bound to another group; insuch cases, the identity of the group is denoted by the group name(whether or not there is an adjacent dash in the text). Note that wherecontext indicates, a single group may be attached to more than one othergroup (e.g., where a linkage is intended, such as linking groups).

“Bound” may be used herein to indicate direct or indirect attachment. Inthe context of chemical structures, “bound” (or “bonded”) may refer tothe existence of a chemical bond directly joining two moieties orindirectly joining two moieties (e.g. via a linking group or any otherintervening portion of the molecule). The chemical bond may be acovalent bond, an ionic bond, a coordination complex, hydrogen bonding,van der Waals interactions, or hydrophobic stacking, or may exhibitcharacteristics of multiple types of chemical bonds. In certaininstances, “bound” includes embodiments where the attachment is directand also embodiments where the attachment is indirect. “Free,” as usedin the context of a moiety that is free, indicates that the moiety isavailable to react with or be contacted by other components of thesolution in which the moiety is a part.

When any range of numbers is mentioned herein, every number within therange (particularly every whole number) is specifically included herein.For example, a range of 1 to 14 C atoms specifically includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 C atoms.

DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings, in which:

FIG. 1: RP-HPLC chromatogram of a TT dimer phosphorothioate and a TTdimer phosphate.

FIG. 2: Total Ion Chroamtogram (TIC) obtained from Ion Trap LC/massspectrometry analysis of a 13-mer DNA phosphorothioate thioalated with0.2M PADS in propylene carbonate/NMI:90/10:v/v.

FIGS. 3 a and 3 b: Ion Trap LC/MS chromatograms of a 13-mer DNAphosphorothioate thioalated with 0.2M PADS in propylenecarbonate/NMI:90/10:v/v. From these chromatograms and tabulated data, itis possible to determine the total amount (in %) of P═O oligonucleotidethat contaminates the 13-mer DNA phosphorothioate oligonucleotidethioalated with 0.2M PADS in propylene carbonate/NMI:90/10:v/v.

FIG. 4: Comparison of a TT dimer thiolation efficiency performed with a0.2M PADS solution prepared with different solvent in the presence (orabsence) of 10% N-methyl imidzaole (v/v).

FIG. 5: Comparison of TT dimer thiolation efficiency performed with 0.2MPADS in different solvents (no N-alkyl imidazole).

FIGS. 6 a and 6 b: Comparison of TT dimer thiolation efficiencyperformed with 0.2M PADS in various toluene/N-methyl imidazole solutionsfreshly prepared (6 a) or aged for 24 hrs (6 b).

FIGS. 7 a and 7 b: TT Comparison of dimer thiolation efficiencyperformed with 0.2M PADS in various MeCN/N-methyl imidazole solutionsfreshly prepared (7 a) or aged for 24 hrs (7 b).

FIGS. 8 a and 8 b: Comparison of TT dimer thiolation efficiencyperformed with 0.2M PADS in various 2-Methyl THF/N-methyl imidazolesolutions freshly prepared (8 a) or aged for 24 hrs (8 b).

DETAILED DESCRIPTION

The following examples illustrate the synthesis of some compounds of thepresent invention, and are not intended to limit the scope of theinvention set forth in the claims appended hereto.

A method of producing a phosphorothioate or a phosphorodithioateinternucleotide linkage into an oligonucleotide, is provided. In someembodiments, the method comprises:

-   -   providing a phosphite or a thiophosphite intermediate    -   contacting said phosphite or thiophosphite intermediate with a        composition comprising an acetyl disulfide, a solvent and a        N-alkyl imidazole for a time sufficient to convert said        phosphite or thiophosphite intermediate to said phosphorothioate        or phosphorodithioate internucleotide linkage.

In certain embodiments, the method of producing a phosphorothioate or aphosphorodithioate linkage in an oligonucleotide, comprises:

-   -   providing a phosphite or a thiophosphite intermediate    -   contacting said phosphite or thiophosphite intermediate with a        composition comprising phenylacetyl disulfide, a solvent and        N-methyl imidazole for a time sufficient to convert said        phosphite or thiophosphite intermediate to said phosphorothioate        or phosphorodithioate internucleotide linkage.

In other embodiments of the method described above, the solvent has alow dielectric constant.

In particular embodiments, the solvent comprises toluene, xylene,2-methyl THF, cyclopentyl methyl ether, acetonitrile or propylenecarbonate.

In embodiments of the invention the solvent may make up at least 30%,50%, 60%, 80%, 90% or even 95% of the composition (in volume) used forcontacting the phosphite or thiophosphite.

In particular embodiment of the described above sulfurization method,the solvent is toluene.

In another embodiment of the sulfurization method, the solvent is2-methyl THF.

In some embodiments of the invention, the method of converting aphosphite or thiophosphite internucleotide linkage of an oligonucleotideto a phosphorothioate or phosphorodithioate is described wherein saidN-alkyl imidazole is at least 1% in volume (v/v) in said solvent (or atleast 5% or at least 10% (v/v) in said solvent). The N-alkyl imidazolemay be up to 10%, 15%, or even 25% (v/v) in said solvent in someembodiments.

In some embodiments, the method of sulfurization features N-alkylimidazole at a concentration of at least 5% in volume (v/v) in saidsolvent.

In particular embodiments, the method of sulfurization is describedwherein said acetyl disulfide is at least at 0.1M concentration in saidsolvent.

In certain embodiments of the of the sulfurization method describedabove, the phenylacetyl disulfide is at a concentration of at least 0.1M and N-methyl imidazole is at least 5% in volume (v/v) in said solventsystem.

In particular embodiments of the sulfurization method described above,the solvent system is toluene, phenylacetyl disulfide is at aconcentration of at least 0.1 M and N-methyl imidazole is at least 5% involume in toluene (v/v).

In particular embodiments of the sulfurization method described above,the solvent system is toluene, phenylacetyl disulfide reagent is at aconcentration of 0.2 M and N-methyl imidazole is 10% to 20% in toluene(v/v).

In particular embodiments of the sulfurization method described abovethe solvent is 2-methyl THF, phenylacetyl disulfide is at aconcentration of at least 0.1 M and N-methyl imidazole is at least 5% involume in 2-methyl THF (v/v).

In particular embodiments of the sulfurization method described above,the solvent system is 2-methyl THF, phenylacetyl disulfide is at aconcentration of 0.2 M and N-methyl imidazole is 10% to 20% in volume in2-methyl THF (v/v).

In certain embodiments, a method of sulfurizing at least one phosphiteor thiophosphite linkage intermediate in an oligonucleotide isdescribed, said method comprising:

-   -   providing a phosphite or a thiophosphite intermediate bound to a        solid support,    -   contacting said phosphite or thiophosphite intermediate with a        composition comprising an acetyl disulfide, a solvent and        N-alkyl imidazole for a time sufficient to convert said        phosphite or thiophosphite intermediate to said phosphorothioate        or phosphorodithioate.

In particular embodiments of the sulfurization method, the solid supportis controlled pore glass or polystyrene.

In particular embodiments of said sulfurization method, the solidsupport is an array substrate or beads.

In some embodiments of the method further comprising a phosphite orthiophosphite intermediate attached to a solid support, the solvent isselected from toluene, xylene, 2-methyl THF, cyclopentyl methyl ether,acetonitrile and propylene carbonate.

In certain embodiments of the invention, the method is featured whereinN-methyl imidazole is at least at 5% in volume and phenylacetyldisulfide at least at 0.1 M concentration in said solvent.

In some embodiments of the sulfurization method described above, theoligonucleotide comprises one or more ribonucleotide.

In certain embodiments of the sulfurization method described above, theoligonucleotide comprises at least one nucleotide analogue.

A composition to thiolate a phosphite or thiophophite linkageintermediate in an oligonucleotide is provided. The compositioncomprises: phenylacetyl disulfide at a concentration of at least 0.1Mand N-methyl imidazole at least at 5% in a solvent (v/v).

In particular embodiments, the composition comprises: toluene as asolvent, phenylacetyl disulfide at a 0.2 M concentration and N-methylimidazole at 10% in toluene (v/v).

In particular embodiments, the composition comprises 2-methyl THF as asolvent, phenylacetyl disulfide at 0.2 M concentration andN-methylimidazole at 10% in 2-methyl THF (v/v).

An oligonucleotide containing at least one phosphorothioate orphosphorodithioate linkage produced by the method of the invention.

Oliognucleotide Synthesis.

The conventional sequence used to prepare an oligonucleotide usingphosphoramidite chemistry basically follows the following steps(Matteucci, M. D., Caruthers, M. H. J. Am. Chem. Soc., 103, (1981),3186-3191): (a) coupling a selected nucleoside through a phosphite orthiophosphite linkage to a functionalized support in the firstiteration, or to the 2′,3′, or 5′ positions of a nucleoside bound to thesubstrate (i.e. the nucleoside-modified substrate) in subsequentiterations (step (a) is sometimes referenced as “phosphitylating”); (b)optionally, but preferably, blocking unreacted hydroxyl groups on thesubstrate bound nucleoside; (c) oxidizing the phosphite linkage of step(a) to form a phosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thefunctionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, in aknown manner.

The foregoing methods of preparing polynucleotides are described indetail, for example, in Caruthers, Science 230: 281-285, 1985; Itakuraet al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310:105-110, 1984; and in “Synthesis of Oligonucleotide Derivatives inDesign and Targeted Reaction of Oligonucleotide Derivatives, CRC Press,Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat.No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP0294196, and elsewhere The phosphoramidite and phosphite triesterapproaches are most broadly used, but other approaches include thephosphodiester approach, the phosphotriester approach and theH-phosphonate approach. The foregoing references and all otherreferences cited in this application area incorporated herein byreference, except insofar as they may conflict with anything in thepresent application.

Embodiments of methods of the present invention can use the conventionalsequence described above except the oxidizing step of step (c) isreplaced with the sulfurization step described herein.

Sulfurization of Oligonucleotides

Most of the known sulfurization reagents, have different drawbacks intheir utilization with respect to 1) ability to transfer the sulfur inexcellent yields, and thus minimizing the formation of P═O units(measured by P═S/P═O ratio) 2) good solubility of the sulfurizationreagent 3) stability of the solutions employed to carry thesulfurization 4) time dependency on optimum activity of the solutions(aging process) 5) short reaction times 6) no side reactions with otherparts of the molecule, and finally 7) cost of the reagent and easyavailability. The reaction of tri-valent phosphorus P(III) with carbonyldisulfides sulfur transfer reagents has long been known to be limited bythe dielectric constant of the solvent that is utilized; this wasspecifically described by Vu, H. and Hirschbein, B. L. (1991)Tetrahedron Lett., 32, 3005-3008. It is this property that has madeacetonitrile (dielectric constant≈37) the most obvious choice ofsolvents for these reactions. The reaction of carbonyl disulfide isproposed to occur by a 2-step mechanism in which the first step is anattack of the lone pair of electrons of the tri-valent phosphorus on oneof the sulfur atoms of the disulfide. This produces an intermediate inwhich the phosphorus atom caries a positive charge, which is stabilizedby the use of a high dielectric constant solvent and allow for theequilibrium to more favor this product. The second step of the reactionis the attack of the thio-acid released in the first step of thisreaction, on the carbonyl groups of the positively charged phosphorusintermediate producing the desired pentavalent phosphorus P(IV) compoundcontaining a non-bridging sulfur atom. To utilize this reaction in alower dielectric constant solvent such as xylene, toluene (dielectricconstant≈3) or 2-methyl-tetrahydrofuran (dielectric constant≈7) anacylation catalyst is utilized to speed up the second step of thereaction and possibly increase the overall yield of the reaction. It isnoteworthy to point out that the acylation catalyst may also increasethe efficiency of this reaction in high dielectric constant solventslike acetonitrile, dimethylsulfoxide (DMSO; dielectric constant≈42), orpropylene carbonate (dielectric constant≈64).

There are essentially two types of acylation catalysts, Lewis-Acidcatalysts and nucleophilic catalysts. In a first initial screen, it wasfound that the best effects in increasing the yield of thesulfur-transfer reaction in low dielectric constant solvents wasobtained using nucleophillic catalysts such as dimethylaminopyridine(DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole (NMI),phosphabicylooctane (PBO), 4-pyrrolodinopyridine (4-PDP),1,5,7-Triazabicyclo[4.4.0]dec-5-ene (DEC) and the like. Of the catalyststhat were tested, NMI gave the best results and offers some advantagesover the other reagents. NMI is a well-known characterized reagenttypically utilized in oligonucleotide synthesis for capping theun-reacted hydroxyl groups with acetic anhydride after coupling of thephosphoramidite reagent to the growing chain of the oligonucleotide.Additionally, NMI has the broadest spectrum of solubility in desirablesolvents such as toluene, 2-methyl-THF, 1-methylpyrrolidinone (NMP), andacetonitrile. The addition of NMI to any of these solvents had aremarkable effect in the sulfur-transfer reaction. In the presence oflow amounts of NMI such as 10% by volume or less, and in solvents withlow dielectric constants, previously deemed not suited for thesulfurization reaction, the conversion of the phosphite internucleotidelinkage to phosphorothioate linkages was obtained with the same orgreater efficiency as compared to previously reported conditions in highdielectric constant solvents such as acetonitrile. This effect was mostremarkably seen using phenyl-diacetyldisulfide (PADS). A 0.2 M solutionof PADS in acetonitrile gave an average of 93.4% conversion of aphosphite triester P(OR)₃ internucleotide bond to the correspondingphosphorothioate triester PS(OR)₃ when reacted for 2 minutes on solidsupport using a 100 fold excess of the PADS reagent as compared to theinternucleotide bond. Under the same conditions, a 0.2 M solution ofPADS in toluene gave only a 7.2% conversion to the phosphorothioatetriester PS(OR)₃. The preferred conditions for using PADS described inthe prior art are PADS at 0.2M concentration in a 50/50 (v/v) solutionof acetonitrile and the highly toxic aromatic amine; 3-picoline. Underthese conditions, a freshly prepared solution of 0.2M PADS in 50/50(v/v) acetonitrile and 3-picoline gave 99.6% conversion to the desiredphosphorothioate triester. However, if 10% in volume of NMI is added toa freshly prepared toluene solution of 0.2 M PADS, the conversion to thephosphorothioate triester drastically increases from 7.2% to 99.7%. Thisremarkable result was repeated with a variety of solvents and in allcases, the addition of an acylation catalyst like NMI allows for the useof low to moderate dielectric constant solvents like toluene (dielectricconstant 2.4), xylene (dielectric constant 2.4), dichloromethane(dielectric constant 9), 1-methylpyrrolidinone (dielectric constant˜30),cyclopentyl methyl ether (dielectric constant 4.7), or 2-methyltetrahydrofuran (2-Me-THF; dielectric constant 6.9), and givescomparable results to the use of high dielectric constant solvents likeacetonitrile and propylene carbonate (FIG. 4). This discovery representsa significant industrial process improvement for the chemical synthesisof oligonucleotides containing sulfur substitutions on theinternucleotide phosphodiester bonds.

It is important to note that even very small increases in the percentageof internucleotide bonds that are sulfurized at each step can havedrastic and significant impact on the overall sulfurization of thedesired oligonucleotide product. For clinical application it isdesirable to maximize the overall sulfurization of the molecule (PS) andminimize the oxidation (PO). For oligonucleotides used in clinicalapplications, a typical sequence can have at least 20 sulfurizedinternucleotide bonds. The overall effective sulfurization of themolecule is calculated by taking the single step sulfurization yield andraising it to the power of the number sulfurized internucleotide bonds.As an example a 99.0% stepwise sulfurization for 20 internucleotidebonds results in an overall sulfurization efficiency of 82%; whereas, a99.8% stepwise sulfurization for 20 internucleotide bonds results in anoverall sulfurization efficiency of 96%. In this example there is asignificant and important difference between an oligonucleotide that 82%sulfurized internucleotide bonds and an oligonucleotide that has 96%sulfurized internucleotide bonds; this difference can have a significantclinical impact.

Recently, further optimization of the use of PADS for oligonucleotidesynthesis was described by Kortz, et. al., (2004) Org. Proc. Res. Dev.,8 (6) 852-858. In this manuscript the authors, describe that the optimumconditions for sulfur transfer is a 0.2M solution of PADS in 50/50: v/vacetonitrile and 3-picoline that has been aged for at least 24 hours.The authors report that a freshly prepared solution of PADS under theseconditions gives 99.5 to 99.7%% conversion to the desiredphosphorothioate triester. However, if the solution is prepared and letat room temperature for 24 hours or greater in other words “aged”), theaged solution gives 99.9% conversion to the desired phosphorothioatetriester. The authors explain the better results by the formation ofpolysulfides that are more reactive than the initial disulfide.

The aging of a sulfurization reagent for 24 hours or greater to achieveoptimum utility is highly undesirable, especially under GMPmanufacturing conditions. The aging process is difficult to control anddefine and needs to be tested and defined prior to utilization. As aclear improvement over the previous protocols, the use of NMI in highdielectric constant solvents like acetonitrile or propylene carbonateprovides that a freshly prepared solution PADS can achieve comparableconversion to the desired phosphorothioate triester as an aged solution(PADS in toluene or acetonitrile). A 0.2 M solution of PADS with 10% NMI(by volume) gave an average of 99.8% or greater conversion inacetonitrile, or in propylene carbonate. Remarkably, a fresh solution ofPADS in 2-methyl THF with 10% NMI also gave a 99.8% conversion to thedesired phosphorothioate triester and in all cases aged solutions ofPADS containing 10% NMI outperform aged solutions not containing NMI.

The synthetic methods described herein may be conducted on a solidsupport having a surface to which chemical entities may bind. In someembodiments, multiple oligonucleotides being synthesized are attached,directly or indirectly, to the same solid support and may form part ofan array. An “array” is a collection of separate molecules of knownmonomeric sequence each arranged in a spatially defined and a physicallyaddressable manner, such that the location of each sequence is known.The number of molecules, or “features,” that can be contained on anarray will largely be determined by the surface area of the substrate,the size of a feature and the spacing between features, wherein thearray surface may or may not comprise a local background regionrepresented by non-feature area. Arrays can have densities of up toseveral hundred thousand or more features per cm2, such as 2,500 to200,000 features/cm2. The features may or may not be covalently bondedto the substrate. An “array,” or “chemical array' used interchangeablyincludes any one-dimensional, two-dimensional or substantiallytwo-dimensional (as well as a three-dimensional) arrangement ofaddressable regions bearing a particular chemical moiety or moieties(such as ligands, e.g., biopolymers such as polynucleotide oroligonucleotide sequences (nucleic acids), polypeptides (e.g.,proteins), carbohydrates, lipids, etc.) associated with that region. Anarray is “addressable” when it has multiple regions of differentmoieties (e.g., different polynucleotide sequences) such that a region(i.e., a “feature” or “spot” or “well” of the array) at a particularpredetermined location (i.e., an “address”) on the array will detect aparticular target or class of targets (although a feature mayincidentally detect non-targets of that feature). Array features aretypically, but need not be, separated by intervening spaces. An array ofpolynucleotides, as described herein, may include a two or threedimensional array of beads. In certain cases, the beads are linked to anoligonucleotide that has two portions, a first portion that binds to atarget, and a second portion that contains a nucleotide sequence thatidentifies the oligonucleotide. In other cases, the bead may provide anoptical address for the oligonucleotide, thereby allowing the identityof the oligonucleotide to be determined. The array may be in the form ofa 3-dimensional multiwall array such as the Illumina BeadChip. Oneembodiment of BeadChip technology is the attachment of oligonucleotidesto silica beads. The beads are then randomly deposited into wells on asubstrate (for example, a glass slide). The resultant array is decodedto determine which oligonucleotide-bead combination is in which well.The arrays may be used for a number of applications, including geneexpression analysis and genotyping. The address is a unique sequence toallow unambiguous identification of the oligonucleotide after it hasbeen deposited on the array. Bead Arrays may have, for example, 1,000 to1,000,000 or more unique oligonucleotides. Each oligonucleotide may besynthesized in a large batch using standard technologies. Theoligonucleotides may then be attached to the surface of a silica bead,for example a 1-5-micron bead. Each bead may have only one type ofoligonucleotide attached to it, but have hundreds of thousands of copiesof the oligonucleotide. Standard lithographic techniques may be used tocreate a honeycomb pattern of wells on the surface, for example a glassslide. Each well may hold a bead. The beads for a given array may bemixed in equal amounts and deposited on the slide surface, to occupy thewells in a random distribution. Each bead may be represented by, forexample, about 20 instances within the array. The identity of each beadmay be determined by decoding using the address sequence. A unique arraylayout file may then associated with each array and used to decode thedata during scanning of the array.

In some embodiments, oligonucleotides being synthesized may be attachedto a solid support (for example: beads, membrane, 96-well plate, arraysubstrate, filter paper and the like) directly or indirectly. Suitablesolid supports may have a variety of forms and compositions and derivefrom naturally occurring materials, naturally occurring materials thathave been synthetically modified, or synthetic materials. Examples ofsuitable support materials include, but are not limited to, control poreglass (CPG), silicas, teflons, glasses, polysaccharides such ascellulose, nitrocellulose, agarose (e.g., Sepharose (from Pharmacia) anddextran (e.g., Sephadex and Sephacyl, also from Pharmacia),polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers ofhydroxyethyl methacrylate and methyl methacrylate, and the like. Theinitial monomer of the oligonucleotide to be synthesized on the solidsupport, e.g. CPG, bead, or array substrate surface, can be bound to alinking moiety (for example, a succinyl linker,or a hydroquinone—O,O′-diacidic acid called a “Q-linker”, an oxalyl linker, and the like)which is in turn bound to a surface hydrophilic group, e.g., a surfaceamine or a hydroxyl present on a silica substrate. In some embodiments,a universal linker is used (for example, Unylinker which is a succinylderivative of8,9-Dihydroxy-4-phenyl-10-oxa-4-aza-tricyclo[5.2.1.02,6]decane-3,5-dione,or other Glenn Research universal supports). In some embodiments, aninitial nucleotide monomer is reacted directly with a reactive site,e.g. a surface amine or hydroxyl present on the substrate. In someembodiments wherein the initial nucleotide monomer is reacted directlywith the reactive sites on the surface, the oligonucleotide remainscovalently attached to the surface post oligonucleotide synthesis anddeprotection, after all of the protecting groups are removed. In someembodiments, a nucleotide monomer is reacted with a non-nucleosidehydroxyl or amine that is not part of a nucleoside or nucleotide.Alternatively, in some embodiments, an oligonucleotide can besynthesized first and then attached to a solid substrate post-synthesisby any suitable method. Thus, particular embodiments can be used toprepare an array of oligonucleotides wherein the oligonucleotides areeither synthesized on the array,or attached to the array substratepost-synthesis.

EXAMPLES Synthesis of TT Dimers (General Procedure).

All DNA phosphorothioate dimers syntheses were performed on 1 mmolescale using thymidine attached to controlled pore glass beads (CPG) by asuccinic acid linker or a hydroquinone linker (Q-linker), both of whichare commercially available columns from Glen Research, Sterling Va. Forthe purpose of the experiments, the standard 1 mmole cyanoethyl cyclewas modified by replacing capping step with thiolation on an AppliedBiosystems model 394 DNA/RNA synthesizer, Foster City, Calif. Afterstandard 25 sec coupling, CPG was washed with MeCN and dried with argon.Thiolating solution (placed in bottle 11) was then delivered to thecolumn for 15 sec, and let sit for 2 minutes. After that time, CPG waswashed with DCM, folllowed by MeCN, and the cycle continued withstandard I₂ oxidation (to ensure complete conversions all P-IIIphosphites into stable P-V triesters—in case not all the linkages wereoxidized with sulfur) and final detritylation.

Cleavage from the support and deprotection were done in gas ammonia (70psi) at RT for 2.5 hours. After venting the ammonia gas, crude productswere dissolved in water (1 mL), filtered and analyzed by RP-HPLC(ODS-Hypersil column, 5 mm 4.0×250 mm, 0.1M TEAA buffer with MeCNgradient (0-20% in 40 minutes). The ratio of phosphodiester tophosphorothioatediester was determined by peak integration (FIG. 1) onan Agilent Technologies, model 1200 High Performance LiquidChromatography (HPLC) System, Santa Clara, Calif.

I. PADS Thiolation Efficiency in Different Solvents without the Use ofN-methyl imidazole

A series of experiments were carried out to evaluate the efficiency ofPADS thiolation of a TT dimer in different solvent systems and withoutthe use of N-methyl imidazole as an acylation catalyst.

The efficiency of thiolation was calculated as explained above bydetermining the ratio of phosphodiester amount tophosphorothioatediester amount extrapolated by peak area integration ofthe phosphodiester and phosphorothioate species from the HPLCchromatogram.

The results are summarized in the FIG. 5.

II. Titration of NMI in 0.2M PADS Solutions.

A two set of series of experiments were carried out to determine theoptimal amount of acylation catalysts (NMI) needed to give the bestthiolation results with PADS at 0.2M in different solvent systems. Thefirst set was performed with freshly made PADS solutions and the secondset was performed with 24 hrs aged solutions. The efficiency ofsulfurization of the phosphite intermediate to a phosphorothioatelinkage of a TT-dimer was calculated as described previously, bydetermining the ratio of phosphate diester to phosphorothioate diesterfrom the HPLC chromatogram. The results are summarized in FIGS. 6 a-8b).

III. Synthesis of Test Phosphorothioate 13-mer (all DNA) with VariousPADS Formulations.

Instrumentation: GE Akta Oligopilot 100

Synthesis Details: 5′ gCA TTg gTA TTC T 3′ synthesized at 490 μMolsynthesis scale on GE PS200 solid support loaded at 204 μMol/g with 2′deoxy Thymidine

Standard oligonucleotide synthesis cycle:

Detritylation

-   3% Dichloroacetic Acid in Dichloromethane

Coupling

-   0.2M phosphoramidites in acetonitrile-   0.5M EthylthioTetrazole in acetonitrile-   2eq. of monomer (relative to synthesis scale)-   3.7:1 ethylthiotetrazole (ETT:monomer molar ratio)-   Recycled for 7 minutes

Oxidation/Thiolation

-   3.92eq. of thiolation reagent (relative to synthesis scale)-   ˜12 minutes total contact time

Thiolation Solutions Utilized

0.2M PADS in Propylene Carbonate/10% N-Methyl Imidazole, freshlyprepared

0.2M PADS in Toluene/10% N-Methyl Imidazole, freshly prepared

0.2M PADS in Toluene/10% N-Methyl Imidazole, ˜24 hours aged

0.2M PADS in Acetonitrile/3-Picoline (1:1), freshly prepared

0.2M PADS in Acetonitrile/3-Picoline (1:1), ˜24 hours aged

Capping

-   0.5 column volume each reagent, delivered simultaneously-   20% n-Methyl Imidazole in Acetonitrile-   5:3:2 Acetonitrile:2,6-Lutidine:Acetic Anhydride

Cleavage and Deprotection

Solid support samples of ˜100 mg were deprotected in 1 ml aqueous MethylAmine for 1 hour at 40° C., cooled, filtered & diluted with deionizedwater.

Analysis

Analysis performed on Agilent 6330 Ion Trap LC/MS

Tabulated mass-spec data from Thiolation synthesis experimentsCalculated stepwise efficiency PADS Solutions P = S % P = O % (%) 0.2MPADS in Propylene Carbonate/ 94.5 5.5 99.53 N-Methyl Imidazole, 90/10,v/v, freshly prepared 0.2M PADS in Toluene/N-Methyl 96.4 3.6 99.69Imidazole, 90/10, v/v, freshly prepared 0.2M PADS in Toluene/N-Methyl97.4 2.6 99.78 Imidazole, 90/10, v/v, ~24 hours aged 0.2M PADS inAcetonitrile/3-Picoline 94.5 5.5 99.53 (1:1), freshly prepared 0.2M PADSin Acetonitrile/3-Picoline 97.4 2.6 99.78 (1:1), ~24 hrs

Efficiency Calculation

There are 12 thiolation reactions in the test molecule (13 nucleosides),so the stepwise efficiency can be calculated by the following formula.

$\text{?}\sqrt{12}$?indicates text missing or illegible when filed                    

Example:

${\text{?}\sqrt{12}} = {0.9978 = {99.78\%}}$?indicates text missing or illegible when filed                    

Ion Trap LC/MS Data (FIGS. 3 a and 3 b) of a_(—)13-mer phosphorothioateDNA sulfurized with 0.2M PADS in Propylene Carbonate/NMI Fresh Solution.

LC Buffer: 5 mM NH4OAc/MeOH.

From the mass spectrometry chromatogram and the quantitative dataassociated with it, it is possible to determine the total amount of P═O(expressed in %) in the sample. From these data, it is possible thus todetermine the total amount of P═S in the same sample and to calculatethe average stepwise sulfurization efficiency using the formula as shownpreviously.

For example, in the m/z data obtained from the mass spectrum ofphosphorothioate 13-mer DNA thiolated with 0.2 M PADS in propylenecarbonate/NMI: 90/10 (v/v)) fresh solution (chromatogram shown in FIG. 3b), the total P═O amount is given as 5.5%, thus total P═S is calculatedas 94.5%, and the average stepwise thiolation efficiency is calculatedas 0.945^((1/12))=0.9953 or 99.53%.

ABBREVIATIONS

In this application, the following abbreviations have the followingmeanings.

Abbreviations not defined have their generally accepted meanings.

-   ° C.=degree Celsius-   hr=hour-   min=minute-   sec=second-   μM=micromolar-   mM=millimolar-   M=molar-   ml=milliliter-   μl=microliter-   mg=milligram-   μg=microgram-   v/v=volume/volume-   DMAP=4,4′-dimithylaminopyridine-   DMT=4,4′-dimethoxytrityl-   DCM=Dichloromethane-   MeCN=Acetonitrile-   2-Me-THF=2-Methyl-tetrahyfdrofuran-   NMI=N-methyl Imidazole-   Py=Pyridine-   Pic=3-Picoline-   TEA=triethylamine-   TEAA=triethylammonium acetate-   TEAB=triethylammonium bicarbonate-   TEMED=N,N,N′,N′-tetramethylethylenediamine-   TBAF=tetrabutylammonium fluoride-   TBDMS=tert-butyl-dimethylsilyl-   RP-HPLC=Reverse Phase High Performance Liquid Chromatography-   RT=Room Temperature

One or more embodiments of the invention may provide for one or more, orother, useful aspects such as now discussed. However, these are notlimiting of the invention nor any particular embodiment. Chemicalsynthesis of biologically important alkyl phosphite products and thechemical entities useful in such synthesis, and oxidative addition ofsulfur to such phosphites intermediates. The formation of P=S linkagesin biologically important compounds may be allowed, without the need forsolvents with high dielectric constants. Sulfur addition according toone or more embodiments may take place with high yields, in either thecurrent industrially preferred large-scale solvents or solvents that arederived from renewable resources. The renewable solvents have asignificantly lower carbon footprint than the current industrialpetrochemical based solvents which may be prone to production shortagesand price volatility. The thiolation reaction may also be significantlyless expensive, and may give equally high or higher efficiency of sulfurtransfer than methods that use industrially non-preferred solvents.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itis readily apparent to those of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modificationsmay be made thereto without departing from the spirit or scope of theappended claims.

Accordingly, the preceding merely illustrates the principles ofembodiments of the invention. It will be appreciated that those skilledin the art will be able to devise various arrangements which, althoughnot explicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the invention aswell as specific examples thereof, are intended to encompass bothstructural and functional equivalents thereof. Additionally, it isintended that such equivalents include both currently known equivalentsand equivalents developed in the future, i.e., any elements developedthat perform the same function, regardless of structure. The scope ofthe present invention, therefore, is not intended to be limited to theexemplary embodiments shown and described herein. Rather, the scope andspirit of present invention is embodied by the appended claims.

1. A method of sulfurizing at least one phosphite or thiophosphitelinkage in an oligonucleotide, the method comprising: contacting anoligonucleotide comprising a phosphite or thiophosphite intermediatewith a composition comprising an acetyl disulfide, a solvent, and anucleophilic acylation catalyst, for a time sufficient to convert thephosphite or thiophosphite intermediate to a phosphorothioate orphosphorodithioate; wherein the nucleophilic acylation catalyst is at upto 25% (v/v) in the solvent.
 2. The method of claim 1, wherein thenucleophilic acylation catalyst is an N-alkyl imidazole.
 3. The methodof claim 2, wherein the N-alkyl imidazole is N-methyl imidazole.
 4. Themethod of claim 1, wherein the solvent is at least 80% (v/v) of thecomposition.
 5. The method of claim 2, wherein the N-alkyl imidazole isat 10% (v/v) or less in the solvent.
 6. The method of claim 1, whereinthe solvent is a high dielectric constant solvent.
 7. The method ofclaim 5, wherein said solvent comprises acetonitrile or propylenecarbonate.
 8. The method of claim 7, wherein the solvent isacetonitrile.
 9. The method of claim 1, wherein the solvent is a lowdielectric constant solvent.
 10. The method of claim 9, wherein saidsolvent comprises toluene, xylene, 2-methyl THF, cyclopentyl methylether or 1-methylpyrrolidinone.
 11. The method of claim 10, wherein thesolvent is toluene.
 12. The method of claim 2, wherein the N-alkylimidazole is at least 5% in volume (v/v) in the solvent.
 13. The methodof claim 12, wherein the acetyl disulfide is at least at 0.1Mconcentration in said solvent.
 14. The method of claim 1, wherein theoligonucleotide is bound to a solid support.
 15. The method of claim 14,wherein said solid support is an array or beads.
 16. The method of claim1, wherein the oligonucleotide comprises one or more ribonucleotides.17. The method of claim 1, wherein the oligonucleotide comprises atleast one nucleotide analogue.
 18. A method of sulfurizing at least onephosphite or thiophosphite linkage in an oligonucleotide, the methodcomprising: contacting an oligonucleotide comprising a phosphite orthiophosphite intermediate with a composition comprising an acetyldisulfide, a high dielectric solvent, and N-methyl imidazole, for a timesufficient to convert the phosphite or thiophosphite intermediate to aphosphorothioate or phosphorodithioate; wherein the high dielectricsolvent is at least 80% (v/v) of the composition, and the N-methylimidazole is at 10% (v/v) or less in the high dielectric solvent. 19.The method of claim 18, wherein the solvent is acetonitrile.